Studies on the Cell Wall of Chlorella III. Incorporation of Photosynthetically Fixed Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

The carbon metabolism in cell walls of Chlorella ellipsoideawas studied by following ¹⁴C incorporation into cell wall constituentsin photosynthesizing, synchronously growing cells. The rateof incorporation was higher at an early growth phase of thecell cycle. The ¹⁴C was incorporated into both the major cellwall constituents, hemicellulose and ‘rigid wall’,and the radioactivity in the latter was distributed into itstwo components, glucosamine and amino acids. In pulse-chaseexperiments, the ¹⁴C fixed photosynthetically in the precedingcell cycle was rapidly transferred into the cell wall constituentsat the early growth phase of the ongoing cell cycle, and thereafterwas gradually released from the cell walls, although the totalamount of ¹⁴C in the cells remained constant. It was concludedthat the cell wall constituents are turned over during the growthphase of the algal cell cycle, and that the cell wall metabolismin the ongoing cell cycle is closely connected with the carbonmetabolism in the preceding cell cycle. (Received February 3, 1982; Accepted June 21, 1982)     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. Gibberellin-coumarin and kinetin-coumarin interactions on stem elongation

In azuki bean (Azukia angularis = Vignia angularis) epicotylsections, 5 ? 10^–4 M coumarin inhibited the incorporationof radioactivity from [U^–14C]glucose into the cellulosefraction by 35% in the absence of indole-3-acetic acid (IAA)and by 40% in the presence of 1 ? 10^–4 M IAA. There wasno inhibitory effect on the incorporation of radioactivity intothe other fractions. Coumarin at 5 ? 10^–4 M reversed thepromoting effect of 1 ? 10^–5 M gibberellin A₃ (GA) andthe inhibitory effect of 1 ? 10^–5 M kinetin on IAA-inducedelongation of sections with no significant effects on IAA-inducedelongation. Neither GA nor kinetin had any appreciable effectson cellulose synthesis. No inhibition of cellulose syntheiswas observed with 1 ? 10^–3 M colchichine, which has beenreported to have effects similar to those of coumarin on GA-or kinetin-affected stem elongation. Coumarin at 5 ? 10^–4M was ineffectual in breaking up wall microtubules, while adisrupting effect on wall microtubules was clearly demonstratedwith 3 ? 10^–4M colchicine. From these results, the possible involvement of cellulose synthesisin cell expansion controlled by GA or kinetin was suggested. (Received August 3, 1973; )     

Effects of IAA on oxygen-sensitive growth and on hydroxyproline protein level in cell wall

Indole-3-asscetic acid (IAA) accelerated the incorporation ofradioactivity derived from ¹⁴C-proline into the SLS-insolublecell wall fraction only when the sections were exposed to lowoxygen concentrations. However, IAA showed no effect on theratio of hydroxyproline to proline incorporated into the SLS-insolublefraction in both 20% and 8% O₂-treated sections. The amountof hydroxyproline rigidly bound to the cell wall increased withincreasing IAA concentration in 8% O₂-treated sections, whilethat of the 20% O₂-treated ones decreased with IAA treatment. On the other hand, IAA increased the amount of ¹⁴C-labeled hydroxyprolineincorporated into the SLS-insoluble fraction of sections treatedwith cycloheximide, and their elongation was greatly inhibited. Based on the results that O₂ and IAA affect the auxin-inducedand the oxygen- sensitive growth differently, we suggest thatboth types of growth may antagonize each other in response tochanges in O₂ and IAA concentrations, resulting in balancedgrowth in the cell. (Received October 7, 1977; )     

Effect of {alpha}-Tomatine on the Response of Wheat Coleoptile Segments to Exogenous Indole-3-acetic Acid

A concentration of 10^–5 M tomatine had no effect on leakagefrom, or elongation of, wheat coleoptile segments, but consistentlyreduced IAA-enhanced extension growth by c. 50 per cent. Therewas no evidence of chemical interaction between the alkaloidand the auxin in solution, and IAA action was not affected bypre-treatment for up to 3 h with 10^–5 M tomatine. Studieswith [2-¹⁴C]IAA revealed that 10^–5 M tomatine did notinhibit uptake of auxin into segments. The effect of pre-treatingsegments for up to 3 h with IAA could be virtually nullifiedby 10^–5 M tomatine, as could also IAA-induced changesin properties of coleoptile cell walls. Results are discussedin relation to the ability of tomatine to disrupt membrane functionand to current hypotheses implicating membranes in the primaryaction of auxin.     

Total and Polar Lipid Biosynthesis during Crotalaria juncea Linn. Pollen Tube Growth: Effect of Gibberellic Acid, Indoleacetic Acid and (2-Chloroethyl)-phosphonic Acid

Gibberellic acid (GA₃) at 58 µM, indoleacetic acid (IAA)at 29 µM, and (2-chloroethyl) phosphonic acid (Ethephon)at 70 µM promoted pollen tube growth in Crotalaria junceapollen suspension cultures both in water and basal medium. GA₃stimulated [ l-¹⁴C]acetate incorporation into total lipids inboth media, whereas IAA enhanced incorporation in water culturesonly. On the contrary, Ethephon reduced the label in total lipidswhen supplemented in basal medium. Based on [l-¹⁴C lacetateincorporation into different phospho- and glycolipids, it isproposed that these growth regulators have a definite role inthe biosynthesis of lipid components of the membranes.     

Effect of inhibition of protein glycosylation on auxin-induced growth and the occurrence of osmiophilic particles in maize (Zea mays L.) coleoptiles

The dependence of auxin (IAA)-induced elongation growth on proteinglycosylation was investigated in abraded maize (Zea mays L.)coleoptile segments, employing 2-deoxy-D-glucose (DOG) and tunicamycin(TUM) as inhibitors of protein glycosylation. TUM had no detectableeffect on growth at 100µg ml^–1. DOG impaired growthat concentrations larger than 1 mM. Total inhibition of growthoccurred at a concentration of 20 mM. Similar effects were observedwith mannose and glucosamine. The effect on wall-synthetic processesin the growth-limiting epidermis was analysed by tracer incorporationstudies. Within 30 min hemicellulose and cellulose synthesis,measured as ³H-glucose incorporation, was not affected by DOG,indicating that inhibition of growth is not causally relatedto synthesis of both wall components. In contrast, protein synthesisand secretion into the walls, measured as incorporation of ³H-leucineinto the TCA-precipitable protoplasmic and wall-bound protein,was rapidly inhibited by DOG. Concomitant with the effect ongrowth, DOG as well as mannose inhibited the occurrence of osmiophilicparticles (OPs) which specifically occur at the growth-limitingepidermis during IAA-induced growth. The results provide evidencethat IAA-induced wall loosening underlying elongation growthis dependent on O-glycosylation of proteins and their subsequentsecretion into the epidermal walls. It appears that interferencewith these processes is responsible for inhibition of IAA inducedgrowth by hexoses acting as anti-glucose antimetabolites. Key words: Auxin-induced growth, cell-wall synthesis, 2-deoxy-D-glucose, mannose, osmiophilic particles, tunicamycin     

De novo synthesis and hormonal regulation of a dipeptidase in Cucurbita maxima

The following evidence was obtained for the de novo synthesis of dipeptidase in squash (Cucurbita maxima Duch. var. Hubbard) cotyledons during germination: (i) the amount of [¹⁴C]leucine incorporated into the dipeptidase was greater than that found in other proteins; (ii) the enzyme coincided with a peak of radioactivity in DEAE column chromatography; and (iii) the specific radioactivity of the enzyme increased with purification. There was also a positive correlation between the rate of [¹⁴C]leucine incorporation into dipeptidase and the rate of dipeptidase development. Four plant growth regulators, gibberellic acid (GA) benzyladenine (BA), indol-3-acetic acid (IAA), and abscisic acid (ABA) were examined for their effect on the development of dipeptidase activity at 5 × 10^?6 and 5 × 10^?5 M. None of these regulators affected the activity of the isolated dipeptidase per se. In intact see ds, BA and IAA inhibited the development of dipeptidase activity at the higher concentration, ABA reduced the activity at both concentrations; however, GA enhanced its development at the higher concentration. In distal-half cotyledons, BA and GA stimulated enzyme development but they showed no synergistic effect. IAA suppressed the development of enzyme activity at the higher concentration and ABA inhibited development at both levels.     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 1988)     

The effect of indole-3-acetic acid on coleoptile extension growth in the absence of protein synthesis

Summary In wheat coleoptile sections cycloheximide inhibited over 90% of ¹⁴C-leucine incorporation into protein within 10 minutes of its application. Even after 2-hour pretreatments with cycloheximide, IAA stimulated extension, suggesting that its growth-promoting action did not directly involve protein synthesis.Kinetic experiments with cycloheximide indicate that incorporation of a structural factor (possibly protein from a previously synthesised pool) into cell walls might be the rate-limiting process affected by IAA.     

Turgor Regulation in a Brackish Water Charophyte, Lamprothamnium succinctum III. Changes in Cytoplasmic Free Amino Acids and Sucrose Contents during Turgor Regulation

Cytoplasm and cell sap of Lamprothamnium succinctum were analyzedseparately for the contents of free amino acids and sucroseto find whether they contribute to turgor regulation. In thevacuole, both amino acids and sucrose were found to be minorcomponents contributing to the generation of osmotic pressure.Their amounts were almost insensitive to changes in externalosmotic pressure. In the cytoplasm, both amino acids and sucrosein the cytoplasm contributed about 20% to the osmotic pressure.Hypotonic treatment did not affect the contents of either, buthypertonic treatment, while not affecting the amino acid contents,caused a significant increase in sucrose content. The cytoplasmicsucrose content increased linearly with an increase in externalosmotic pressure, accounting for 40% of the increased osmoticpressure. ¹ Present address: Department of Biology, Osaka Medical College,Sawaragi-cho, Takatsuki, Osaka 569, Japan ² Present address: Department of Applied Physiology, NationalInstitute of Agrobiological Resources, Yatabe, Tsukuda, Ibaragi305, Japan (Received November 25, 1986; Accepted March 18, 1987)     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)     

RIBONUCLEIC ACID METABOLISM AND CELL EXPANSION IN OAT COLEOPTILE

RNA metabolism in oat coleoptiles was studied using physiologicalresponses to 5-FU and actinomycin D; autoradiographic detectionof RNA and protein synthesis; and estimation of ribosomal concentrationby analytical ultracentrifugation. 5-FU failed to inhibit growthof either intact coleoptiles or isolated coleoptile segmentsbut completely blocked cell division in roots. Actinomycin Dmarkedly inhibited auxin-induced expansion of coleoptile segments.When supplied to isolated segments from coleoptiles of variouslengths the RNA precursors cytidine, adenine and adenosine allshowed weak incorporation into RNA of nuclei and in some cases,to a lesser extent, RNA of cytoplasm. IAA did not affect thisRNA synthesis but it was considerably reduced by actinomycinD. A proportion of the label incorporated from RNA precursorswas not removable with either RNase, PCA or hot TCA but wasextracted by trypsin. The amount of this spurious incorporationincreased with coleoptile age, as did the ability to incorporatelabelled amino acids. The concentration of both free and boundribosomes does not increase in growing coleoptiles and may evendecline. Free ribosomes decline markedly in fully grown coleoptileswhile the proportion of bound ribosomes increases. It is concludedthat young coleoptiles contain a full complement of ribosomesnecessary for subsequent growth but normal growth is dependenton continued production of an actinomycin D-sensitive messenger-typeRNA. No evidence for auxin mediation of RNA synthesis was found. ¹Present address: Laboratory of Cell Biology, Faculty of Science,Osaka City University, Sugimoto-cho, Sumiyoshi-ku, Osaka, Japan.     

Apical Dominance in Vicia faba

Apical dominance phenomena have been studied in seedlings ofVicia faba particularly in relation to the movement about theplant of uracil-2-¹⁴C applied to the cotyledons. Decapitationjust below the second node releases the growth of the lowermostlateral bud and inhibition is completely reimposed by applicationof indole-3-acetic acid (IAA) to the cut surface. Uracil-2-¹⁴Capplied in solution to the cotyledons is distributed in thestems of all experimental seedlings with no consistent differencesdue to decapitation or IAA application. On the other hand, decapitationresults in a rapid increase in uracil-2-¹⁴C content in the lateralbuds which far exceeds their promoted growth. This uptake iscompletely suppressed by IAA application. A ring of tri-iodobenzoicacid (TIBA)-lanolin paste around the stem above the bud suppressesIAA action both in bud growth and on uracil-2-¹⁴C uptake, andalso on the movement of IAA-1-¹⁴C down the stem. TIBA-application to the base of the bud does not prevent IAAaction on bud growth, but also does not prevent the movementof IAA-1-¹⁴C (or a water soluble product of its metabolism)into the bud. Direct application of kinetin to the lateral bud of intact plantscauses a short-lived release of growth. Gibberellic acid producesa smaller and scarcely significant increase which is additiveto the kinetin effect. Neither has any effect on uracil-2-¹⁴Cmovement into the bud. The implications of these findings are discussed in relationto various existing theories of the mode of auxin action inapical dominance and it is concluded that their strongest supportis for a mechanism involving the suppression of phloem differentiationin the vascular supply to the bud.     

Effect of Indoleacetic Acid on Growth and Dinitrogen Fixation in Cyanobacteria

The effect of IAA on growth, dinitrogen fixation, and heterocystsfrequency of Anabaena PCC 7119 and Nodularia sp. have been investigated.Concentrations of IAA ranging from 10^–10 to 10^–4M did not change the growth of Anabaena PCC 7119. Concentrationshigher than 10^–4 M were inhibitory. Similar results werefound in Nodularia sp. although in this case the inhibitoryeffect appeared with 10^–5M of IAA. Neither the nitrogenaseactivity nor the heterocysts frequency were enhanced by IAAtreatment. (Received June 17, 1986; Accepted January 22, 1987)     

Effect of Auxin on Autolysis of Cell Walls in Azuki Bean Epicotyls

Native cell walls of azuki bean epicotyls incubated in bufferautolytically released neutral sugars, abundant in galactose,and uronic acids. Treatment with 10^–5 M IAA of subapicalor basal epicotyl segments for 3 h did not influence the amountof total neutral sugars released from the cell walls duringautolysis. However, the amount of glucose and xylose releasedfrom subapical cell walls was increased by IAA. Pretreatmentwith IAA of subapical epicotyl segments enhanced the solubilizationof neutral sugars from pectinase-treated cell walls during incubationin buffer at pH 5 to 6. The amount of fucose, xylose, and glucosereleased was specifically increased by IAA. Of the sugar fractionsreleased from pectinase-treated cell walls during autolysisand subsequently separated by gel filtration on a ToyopearlHW-40S column, IAA promoted the release of oligosaccharides,consisting mainly of glucose and xylose. These results suggestthat autolytic degradation of xyloglucans is closely relatedto IAA-induced growth of azuki bean epicotyls. (Received May 19, 1989; Accepted January 5, 1990)     

The role of cell wall synthesis in sustained auxin-induced growth

The dependence of auxin-induced growth on continued cell wall synthesis was investigated in stem segments of etiolated pea ( Pisum sativum L. cv. Alaska) seedlings using the cell wall synthesis inhibitors monensin and 2,6-dichlorobenzonitrile (DCB). Monensin (5 μ M ) potently inhibited indole-3-acetic acid (IAA)-induced growth, particularly during the second hour of treatment, whereas growth in fusicoccin (FC) was inhibited much less effectively. Incorporation of [¹⁴C]-glucose into both matrix and cellulose fractions of the wall showed a sharp increase beginning after about 60 min, this rise being promoted by both IAA and FC. Monensin inhibited this rise in incorporation of label and completely removed the promotion of this by IAA, although some promotion by FC remained. Monensin inhibited incorporation into cellulose in a manner similar to that into matrix, but the use of the apparently specific cellulose synthesis inhibitor DCB showed that cellulose synthesis could be strongly inhibited without effect on growth, at least in the short term. The results support the view that sustained auxin-induced growth depends upon the incorporation of new matrix cell wall components into the wall.     

Effects of ABA on Synthesis of Cell-Wall Polysaccharides in Segments of Etiolated Squash Hypocotyl I. Changes in Incorporation of Glucose and myo-Inositol into Cell-Wall Components

Externally supplied [³H]myo-inositol and [¹⁴C]glucose were incorporatedin cell-wall fractions of segments of etiolated squash hypocotyl.The extent of incorporation of [¹⁴C]glucose into cell-wall fractionswas very much greater than that of [³H]myo-inositol. Radioactivityfrom [¹⁴C]-glucose was effectively incorporated into hemicelluloseB and cellulose fractions and was incorporated uniformly intohexose, pentose and uronic acid residues, but radioactivityfrom [³H]myo-inositol was incorporated predominantly into uronicacid and pentose residues in the pectin and hemicellulose Bfractions. Exogenously applied ABA significantly suppressed the elongationof segments of squash hypocotyl and the incorporation of radioactivityfrom [^l4C]glucose and [³H]myo-inositol into the segments. Furthermore,ABA significantly inhibited the distribution of incorporatedradioactivity from [¹⁴C]glucose into the cellulose fraction,but did not affect distribution into the pectic fraction. Bycontrast, ABA only slightly inhibited the distribution of theincorporated radioactivity from [³H]myo-inositol into the pecticfraction. These results suggest that most of the cell-wall polysaccharidesin segments of squash hypocotyl are synthesized via the UDP-sugarpathway, and that ABA significantly inhibits the synthesis ofcellulose but not the synthesis of pectic polysaccharides whenABA suppresses the elongation of the segments. (Received March 25, 1988; Accepted November 15, 1988)     

Kinetin Transport to Axillary Buds of Dwarf Pea (Pisum sativum L.)

Decapitation resulted in the transport of significant amountsof ¹⁴C to the axillary buds from either point of application,but pretreatment of the cut internode surface of decapitatedplants with IAA (alone or in combination with unlabelled kinetin)inhibited the transport of label to the axillary buds and resultedin its accumulation in the IAA-treated region of the stem. Inintact plants to which labelled kinetin was applied to the apicalbud there was little movement of ¹⁴C beyond the internode subtendingthis bud; when labelled kinetin was applied to the roots ofintact plants, ¹⁴C accumulated in the stem and apical bud butwas not transported to the axillary buds. A considerable proportionof the applied radioactivity became incorporated into ethanol-insoluble/NaOH-solublecompounds in the apical bud of intact plants, in internodestreated with IAA, and in axillary buds released from dominanceby removal of the apical bud. The results are discussed in relation to the possible role ofhormone-directed transport of cytokinins m the regulation ofaxillary bud growth.     

Cell Wall Metabolism in Developing Strawberry Fruits

Cell wall metabolism was studied in strawberry receptacles (Fragariaananassa, Duchesne) of known age in relation to petal fall (PF).Polysaccharide and protein composition, incorporation of [¹⁴C]glucoseand [¹⁴C]proline by excised tissue, and the fate of ¹⁴CO₂ fixedby young, attached fruits were followed in relation to celldivision, cell expansion, fine structure, and ethylene synthesis. Cell division continued for about 7 d after PF although vacuolationof cells was already beginning at PF and the subsequent cellexpansion was logarithmic. There was an associated logarithmicincrease in sugar content per cell and a decreasing rate ofethylene production per unit fresh weight. During cell expansion radioactivity from [¹⁴C]glucose was incorporatedinto fractions identified as starch and soluble polyuronideand into glucose and galactose residues in the cell wall. Radioactivityfrom [¹⁴C]proline was also incorporated into the cell wall,but only 10 per cent of this activity was found in hydroxyproline.Correspondingly wall protein contained a low proportion of hydroxyprolineresidues. The proportion of radioactivity from ¹⁴CO₂ fixed byfruitlets remained constant in most sugar residues in the cellwall. The proportion of radioactivity in galactose fell, indicatingturnover of these residues. Between 21 and 28 d after PF receptacles became red and softenedbut there was no change in the rate of ethylene production.Cell expansion continued for at least 28 d. Tubular proliferationof the tonoplast and hydration of middle lamella and wall matrixmaterial had begun 7–14 d after PF but became extremeduring ripening. Associated with the hydration of the wall,over 70 per cent of the polyuronide in the wall became freelysoluble, and arabinose and galactose residues lost from thewall appeared in soluble fractions. There was no increase intotal polysaccharide during ripening and incorporation of [¹⁴C]glucoseinto polysaccharides ceased, although protein increased andincorporation of [¹⁴C]proline into wall protein continued.     

The Relationship between Growth and Proline Incorporation after Auxin Treatment of Mung Bean Hypocotyls

Indoleacetic acid (IAA) stimulates the incorporation of ¹⁴C-proline into both the cyloplasmic and the cell wall fractions of the hypocotyl of mung bean (Phaseolus aureus Roxb. cv. Black). It neither stimulates the transfer of ¹⁴C-proline from the cyloplasmic fraction into the cell wall fraction, nor the retention of ¹⁴C-proline in the wall or cytoplasmic fractions. Moreover, the stimulation of growth caused by IAA parallels the stimulation of the incorporation of proline into the cytoplasmic fraction, but does not parallel the stimulation into the cell wall fractions. The stimulation of the incorporation into the cyloplasmic fraction seems to appear within 30 minutes after auxin treatment, at about the same time the increase in the growth is observed in response to IAA, suggesting a connection between these effects. On the other hand, the stimulation of the proline incorporation into the cell wall fraction seems to require more than 90 minutes after auxin treatment, suggesting no close connection between growth and proline incorporation into the cell wall fraction.